for 15 min at room temperature to remove cells and debris. Thrombin was added and samples were incubated for 5 min at room temperature to remove fibrinogen, centrifuged at 10,000 ×for 60 min at 4 °C, centrifuged at 12,000 ×for 5 min, and the resulting pellet was resuspended in 250 ul of sterile phosphate buffered saline 1X, pH 7.4. Samples were aliquoted for quantification of cytokines/chemokines and growth factors.
For each well, we used the median fluorescence intensity of all beads measured for a given analyte and averaged the two replicates and results were accepted when the coefficient of variation was below 15%.]. Samples from the 49 ME/CFS cases and 49 controls included in this study were run in two batches of 20 samples and 78 samples . The 20 samples in the first batch were randomly selected. The cases and controls were frequency-matched on the same matching variables as the total study population.